Study On The Relationship Between 25-hydroxyvitamin D And The Occurrence/Short-term Prognosis Of Systemic Lupus Erythematosus,and Its Regulatory Mechanism

Background and objective:Systemic lupus erythematosus（SLE）is an autoimmune disease involving multiple systems,which is meanly caused by the interaction between genetic and environmental factors.It has long-term duration and alternating relapse-remission characteristics.The irreversible organ damage caused by autoimmune inflammation and treatment side effects are important reasons for poor prognosis,and they are also the focus of current SLE management strategies.25-hydroxyvitamin D（25-OHD）is a fat-soluble vitamin which has the function of bone deposition and immune modulation.Its deficiency has been found to be associated with the onset and severity of SLE,and has been considered as an environmental trigger for SLE and an important means of optimizing SLE treatment strategies.However,the conclusions of up-to-date randomized controlled trials did not support the reduction of autoantibody titers and disease activity by 25-OHD supplementation in SLE patients.25-OHD is still used as an empirical treatment against glucocorticoid（GC）side effects in the clinical management of SLE,and there is no unified expert consensus.Therefore,several research questions remain to be explored regarding the association between25-OHD and SLE:first,whether this correlation is causal,and whether the rationality of 25-OHD supplementation in the prevention and treatment of SLE can be supported by causality;second,whether 25-OHD supplementation can improve the prognosis of SLE patients,and whether the current empirical supplementation strategy needs to be optimized;third,in the treatment of SLE,whether the immune regulatory effect of25-OHD can be fully exerted,and what regulatory mechanisms may affect it.This study used Mendelian randomization,cohort study,network pharmacology and bioinformatics analysis,case-control study,and in vitro experiments,attempting to comprehensively elaborate on the role of 25-OHD in the prevention and treatment of SLE in terms of supplementation rationality,benefits,and optimization methods,making contributions to improving the disease management strategies.Methods:（I）In the first part of this study,based on genome-wide association study（GWAS）data,single nucleotide polymorphisms（SNPs）independently associated with serum 25-OHD levels and SLE were extracted as instrumental variables（IVs）to conduct a bi-directional two-sample Mendelian randomization（MR）study.The bidirectional causal relationships between 25-OHD levels and the risk of SLE onset were inferred.（II）In the second part of this study,based on the SLE registry study database developed by the Chinese SLE research collaboration group,a bi-directional cohort study was conducted.We collected the baseline and regular follow-up data of newly diagnosed SLE patients who visited the First Hospital of Jilin University from 2019 to2023.The follow-up period spanned from baseline to week 52.The SLE patients received Standard of Care（So C）and 25-OHD supplementation at a dosage of1500-2000 IU/d,and monthly follow-up was conducted as planned.Relevant clinical information including the serum 25-OHD level and bone mineral density（BMD）T-value was collected at baseline and follow-up.The SLE disease activity index（SLEDAI）was calculated,and the achievement of SLE responder index for response-4（SRI-4）and modified lupus low disease activity state（m LLDAS）were assessed at every follow-up.The glucocorticoid toxicity index（GTI）was calculated at the week 12 and week 52.2.1 Cox proportional hazard model was used to explore the association between baseline 25-OHD levels,and the achievement of SRI-4 response and m LLDAS during follow-up.Spearman’s rank correlation coefficients were used to explore the relationship between the effect of 25-OHD supplementation,and changes of SLEDAI and ds-DNA antibody titers,to assess the association between the 25-OHD supplement and disease remission;2.2 Generalized additive mixed model（GAMM）was used to fit repeated measurement data,evaluate the effect of 25-OHD supplementation,and analyze the association between baseline/longitudinal 25-OHD levels and changes in BMD T-value/GTI;2.3 Using achieving a normal 25-OHD level（≥20 ng/m L）at week 52 as the outcome,baseline characteristics were used to establish a clinical predictive model for predicting the effect of 25-OHD supplementation based on logistic regression and LASSO algorithms.A nomogram was drew.ROC analyses,calibration curves,decision curve analyses and clinical impact curve were used to evaluate the model’s discrimination,calibration,and clinical utility.（III）In the third part of this study,the regulatory mechanism of 25-OHD immune regulation in SLE was predicted from the perspective of the gene-nutrient interaction,and was verified by case-control study and in vitro experiments.3.1 Network pharmacology and bioinformatics analysis were carried out based on Super-pred target-prediction tool and GEO database.Differential expression gene（DEG）analysis,GO and KEGG function annotation,and protein-protein interaction（PPI）network were used to screen the targets and signaling pathways of 25-OHD against SLE.The hub targets and signaling pathway were obtained through the Network analysis function of Cytoscape software;3.2 Based on expression quantitative trait loci（e QTL）studies and SLE-related GWAS,a summary data-based Mendelian randomization（SMR）was conducted to identify SLE genetic susceptible gene m RNA expression.HEIDI analysis was used to validate the robustness of the causal association;3.3 The hub targets in 3.1 were integrated analyzed with the SLE genetic susceptible genes in 3.2 using PPI network,functional KEGG annotation,and gene-disease enrichment.The potential interactions between genes（X）and nutrient（25-OHD）and their signaling pathways（Y）were screened;3.4 Case-control study was conducted on newly diagnosed SLE patients and age/gender-matched healthy controls（HC）who visited the First Hospital of Jilin University from 2022 to 2023.Clinical information,SLEDAI,serum 25-OHD levels,and PBMCs were collected.q RT-PCR was used to measure the certain gene X m RNA expression.Logistic regression model was used to estimate the association between25-OHD levels,X m RNA expression,and the risk of SLE onset.In the SLE group,patients with a SLEDAI≥15 were classified as severe SLE.The association between25-OHD levels,X m RNA expression,and the risk of severe SLE was calculated;3.5 The B cell stimulating factor of TNF family（BAFF）and Toll-like receptor 9agonist（Cp G-ODN 2006）were used to establish an activated B cell model（Raji cells）.According to serum 25-OHD levels,three intervention concentration gradients were set up:25-OHD deficiency group（10n M）,normal 25-OHD group（60n M）,and sufficient 25-OHD group（100n M）.q RT-PCR,Western Blot,ELISA,and Co-ip were used to verify the effects of 25-OHD on the expression and modification of certain gene X and hub targets,activation of certain signaling pathway Y,and expression of downstream inflammatory cytokines.After Raji cells were transfected with a certain gene X inhibitor or inhibitor-NC,BAFF,Cp G-ODN 2006,and concentration gradient25-OHD interventions were given.q RT-PCR,Western Blot,ELISA,and Co-ip were used to verify the interaction between 25-OHD and a certain gene X in the activation of a certain signaling pathway Y and the release of inflammatory cytokines.Results:（I）The causal relationship between 25-OHD levels and the risk of SLE onsetMR analysis was conducted based on GWAS of European ancestry.The results showed that serum 25-OHD levels had a negative causality with the risk of SLE onset（OR=0.597,95%CI=0.427-0.835）,while there was no reverse causal association（OR=1.003,95%CI=0.995-1.007）.（II）The relationship between 25-OHD levels and short-term prognosis of SLE and the evaluation of empirical supplementation effect1.Association between baseline 25-OHD levels and prognosis of SLEA total of 291 patients were included in the SLE cohort.The Cox proportional hazard model showed that for every 1ng/m L increase in baseline serum 25-OHD level,during the 52-week follow-up,the SRI-4 response rate increased by 7.8%（HR=1.078,95%CI:1.024-1.135）,and the probability of achieving m LLDAS increased by 6%（HR=1.060,95%CI:1.019-1.102）for SLE patients.GAMM showed that patients with25-OHD deficiency at baseline had more BMD T-value decrease(β_interaction=-0.704,SE=0.126)and GTI increase(β_interaction=21.864,SE=6.164)compared to patients with a normal 25-OHD level at baseline.2.Assessment of the effect of 25-OHD supplementationAt baseline,82.5%of SLE patients were in a state of 25-OHD deficiency.GAMM showed that although empirical 25-OHD supplementation of 1500-2000IU/d could increase the serum 25-OHD level of SLE patients by about 0.867ng/m L/month（β=0.867,SE=0.204）,54.2%of patients were still in a state of 25-OHD deficiency at week 12 after the treatment began,and the proportion was still 38.5%at week 52.3.Association between 25-OHD supplementation and prognosis of SLESpearman’s correlation analysis showed that the increase of serum 25-OHD levels was not associated with the decrease of SLEDAI（ρ=-0.084,95%CI:-0.198～0.031）and the decrease of ds-DNA antibody titer（ρ=-0.077,95%CI:-0.194～0.042）.GAMM showed that with the progress of SLE So C,BMD T-value decreased by 0.124 per month（β=-0.124,SE=0.030）,and GTI score increased by8.257 points per month（β=8.257,SE=0.308）.25-OHD supplementation could reduce the decrease rate of BMD T value(β_interaction=0.032,SE=0.002)and the increase rate of GTI(β_interaction=-0.457,SE=0.027),but it was still not enough to avoid the occurrence of GC toxicity.4.The factors related to the effect of 25-OHD supplementationThe LASSO algorithm and logistic regression showed that both initial GC dosage at baseline（OR=0.962,95%CI:0.930-0.996）and 24-hour urine protein level（OR=0.620,95%CI:0.465-0.826）were negatively related to the outcome.Conversely,for every 1ng/m L increase in the baseline 25-OHD level,the probability of achieving a normal serum 25-OHD level increased by 19.6%（OR=1.196,95%CI:1.081-1.324）.The application of tacrolimus（OR=4.215,95%CI:1.239-14.332）and belimumab（OR=6.097,95%CI:1.658-22.423）significantly increased the probability of achieving normal serum levels.（III）The effect of gene-nutrient interaction on the immunoregulatory role of25-OHD in SLE1.Prediction of the hub targets and signal pathway of 25-OHD against SLEThrough network pharmacology and bioinformatics analysis,172 potential targets of 25-OHD were obtained,4847 DEGs between SLE and HC were identified,and 55 intersection genes of 25-OHD targets and SLE-HC DEGs were screened.The GO annotation items of the intersection genes involved 83 MF items,71 BP items,and 102 CC items;KEGG signaling pathway analysis showed that there were 69signaling pathways,involving NF-κB signaling pathway,TNF signaling pathway,Toll receptor signaling pathway and B cell receptor signaling pathway.Network analysis showed that the hub targets of 25-OHD against SLE were NFKB1 and CHUK（corresponding proteins were p105 and IKKα）,and the hub signaling pathway was the NF-κB signaling pathway.2.The screening of genetic susceptibility genes for SLE and the prediction of gene-nutrient interactionsSMR identified 6 susceptible genes associated with the onset of SLE:C4A,BLK,MMP2,TEF,MAP4K4 and UBE2L3.Integrated analysis of these susceptible genes and the hub targets of 25-OHD showed that UBE2L3 was enriched in the NF-κB signaling pathway along with NFKB1 and CHUK.PPI network showed that UBE2L3was located upstream of the two hub targets,mediating ubiquitination modification of the hub targets and up-regulating NF-κB activation.3.The effects of UBE2L3 and serum 25-OHD levels on the onset of SLE and the risk of severe disease activityIn the case-control study,a total of 22 SLE patients and 22 HC were firstly included.The logistic regression analysis showed that both 25-OHD deficiency（OR=1.12,95%CI:1.04-1.20）and UBE2L3 m RNA expression（OR=1.20,95%CI:1.02-1.41）were associated with the risk of SLE onset.Continued to include the SLE patients to 88 cases.Among the 88 SLE patients,there were 30 severe SLE patients and 58 moderate SLE patients according to SLEDAI.The logistic regression analysis also showed that both 25-OHD deficiency（OR=1.52,95%CI:1.19-1.91）and UBE2L3 m RNA expression（OR=1.27,95%CI:1.04-1.56）were associated with the risk of severe SLE.4.25-OHD could only partially inhibit the activation of NF-κB signaling pathway mediated by UBE2L3qRT-PCR and Western blot indicated that BAFF and Cp G-ODN 2006 could significantly activate NF-κB signaling pathway of Raji cells.25-OHD intervention did not significantly inhibit the m RNA and protein expression of two hub targets and UBE2L3.Co-ip revealed that 25-OHD did not significantly inhibit the ubiquitination modification of hub targets mediated by UBE2L3.25-OHD intervention could inhibit the phosphorylation modification of hub targets,with the extent of phosphorylation inhibition increasing with the concentration of intervention;25-OHD intervention also reduced the degradation of IκBαprotein and the nuclear entry of p65 protein,down-regulated the secretion of TNF-αand IL-6.However,these effects did not align with the inhibitory effect on the phosphorylation of hub target proteins,which was only pronounced in the 25-OHD sufficient group（100n M）but weaker in the deficient（10n M）and normal groups（60n M）.5.UBE2L3 knockdown combined with 25-OHD intervention could further inhibit the activation of NF-κB and inflammatory cytokine secretion.Western blot,co-ip and ELISA indicated that UBE2L3 knockdown could significantly inhibit the ubiquitination modification of hub targets,as well as the NF-κB activation,and down-regulate the secretion of TNF-αand IL-6.The combined effect of UBE2L3 knockdown and 25-OHD intervention could further inhibit the NF-κB activation:at 60n M,25-OHD significantly reduced the degradation of IκBαprotein and the nuclear entry of p65 protein,and down-regulated the secretion of TNF-αand IL-6.Meanwhile,in the group with 100n M,the aforementioned effects showed no significant difference compared to the 60n M group.Conclusions:1.There is a negative causal relationship between serum 25-OHD levels and the risk of SLE onset,and it is reasonable for 25-OHD supplementation from the perspective of SLE prevention and etiological treatment.2.Baseline serum 25-OHD level is positively correlated with the short-term prognosis of SLE,including easier access to disease remission and less treatment-related harm.However,the current empirical 25-OHD supplementation dose,aimed at preventing GC treatment-related harm,is insufficient to improve prognosis.Even after supplementation,patients still have varying degrees of 25-OHD deficiency and GC toxicity accumulation.Therefore,the monitoring of 25-OHD should be strengthened,and supplementation treatment strategies should be further optimized based on patient characteristics and treatment regimen.3.Both 25-OHD and UBE2L3 could act as pathogenic factors that affect the risk and severity of SLE.In the activation of BAFF induced NF-κB signaling pathway,UBE2L3 located at upstream of the pharmacological target of 25-OHD.25-OHD can only partly inhibit the up-regulation of NF-κB signaling pathway activation mediated by UBE2L3.Targeting UBE2L3 or increasing serum 25-OHD level could be more beneficial for the exertion of immune modulation of 25-OHD in SLE.